Porphyromonas gingivalis induces an elevated MMP response in dendritic cells, but responses from cultures of single-cell types do not reflect the local tissue environment. The objective of this study was to measure HagB-induced MMP responses in a transwell co-culture system containing dendritic cells, gingival epithelial (GE) keratinocytes, and CD4+ T-cells. Transwell co-cultures were assembled and treated with or without HagB. Immunoassays were used to determine production of MMP1, MMP7, MMP9, and MMP12 in response to HagB up to 64 h. Control responses were subtracted from HagB-induced responses. A two-way fixed effect ANOVA was fit to log-transformed concentrations and pairwise group comparisons were conducted (p < 0.05). At 64 h, dendritic cells produced elevated MMP1 and MMP9 responses, which were attenuated in the 3-cell co-culture (p < 0.05). There were also significant differences in MMP7 and MMP12 production between single-cell cultures and co-cultures. These results support the need to use multiple cell types in culture models to evaluate a more representative response to proinflammatory agonists. This three-cell transwell co-culture model may help us better understand the inflammatory process in periodontal disease and test novel therapeutic approaches.Matrix metalloproteinases (MMPs) are enzymes involved in periodontal tissue destruction. Hemagglutinin B (HagB) from the periodontal pathogen  Porphyromonas gingivalis. The prolonged inflammatory response to this biofilm results in elevated matrix metalloproteinase (MMP) production, which in turn leads to degradation of collagen fibers and subsequent periodontal attachment and bone loss, and ultimately tooth loss if left untreated. Periodontal disease, a chronic inflammatory disease that causes tissue damage in the oral cavity, affects approximately 46% of U.S. adults . PeriodoThe periodontal inflammatory response is a complex process involving communication between cells and strict regulation of cell activities necessary for host defense . A netwoMatrix metalloproteinases are major enzymes implicated in extracellular matrix degradation and tissue remodeling . MMPs arP. gingivalis [in vivo host response. In this study, we utilized a three-cell transwell co-culture, composed of dendritic cells, gingival epithelial (GE) keratinocytes, and CD4+ T-cells, three cell types that play an important role in the innate immune response in periodontal disease. CD4+ helper T-cells were included in the transwell co-culture because they are the most dominant type of T-cell in the gingiva and have been suggested to play roles in homeostasis and immunopathology [Recently, we reported that dendritic cells produce an enhanced cytokine and MMP response when treated with Hemagglutinin B (HagB), a virulence factor of the periodontal pathogen ngivalis . Becauseathology ,10. Dendathology .P. gingivalis HagB-induced MMP response in a uniquely stacked, three-cell transwell co-culture system containing dendritic cells, GE keratinocytes, and T-cells to the MMP response from single-cell cultures.The transwell co-culture allows for the exchange of extracellular biomarkers from one cell type to stimulate co-cultured cells in an allogeneic system. In this study, we explored the use of immune cells and epithelial cells from different hosts in a three-cell transwell co-culture where the cells do not have physical contact with one another. Our study compared the n = 3, n = 9, n = 3) from 0 to 32 h, while n = 9) at 64 h. These values were determined by subtracting the response to the HagB diluent from the HagB-induced response. This not only eliminates the response of any reaction to the diluent, but also eliminates the cell responses induced by the other cell types in the co-cultures and any baseline MMP production from the cells when no agonist is present. In most cases, MMP production was first observed from 8 to 16 h.The production of MMPs in response to HagB was assessed in a modified three-cell transwell co-culture . The prop < 0.05), while GE keratinocytes and T-cells produced little to no MMP1 and MMP9. This trend of MMP1 and MMP9 production was observed throughout the 64 h, starting at 8 h (p > 0.05) from the no cell control at 64 h (The transwell co-cultures were set up with only one cell type. Each cell type was in its respective tier: Dendritic cells (bottom), GE keratinocytes (middle), and T-cells (top). By 64 h, HagB stimulated single cultures of dendritic cells, GE keratinocytes, and T-cells produced varying concentrations of MMP1, MMP7, MMP9, and MMP12 b. Of theg at 8 h a. There  at 64 h b. Howevep < 0.05). At 64 h, the dendritic cell MMP1 response was attenuated in dendritic cells + GE keratinocytes (p < 0.05) and dendritic cells + GE keratinocytes + T-cells transwell co-cultures (p < 0.05)  b. Dendritic cells were also the main drivers of the MMP9 response to HagB, as all cultures containing dendritic cells had the greatest response . HoweverLike the single-cell cultures above, there was little to no production of MMP7 and MMP12 in the transwell co-cultures in response to HagB, as there was no significant difference from the no cell control at 64 h b. HoweveThere has been little research on characterizing MMP responses of cells grown in three-cell transwell co-culture systems using primary GE keratinocytes from the oral cavity. We used the three-cell transwell co-cultures to elucidate cell-to-cell communication that regulates MMP production induced by proinflammatory agonists such as HagB. Co-cultures produced significantly different MMP responses than single-cell cultures, and cytokine and chemokine responses also differed .In this model, dendritic cells drove MMP production, but the expression of MMPs changed in the presence of GE keratinocytes and T-cells. Using transwell co-cultures, we demonstrated how the MMP responses of three cell types differed in a co-culture environment when exposed to the proinflammatory agonist HagB. This experimental approach highlights the contribution and influence multiple cell types in co-culture can have on the MMP response, which plays a role in the pathogenesis of periodontal tissue destruction. Studying the MMP response in this model provides insight into intra- and inter-cellular regulation of MMPs and the role of multiple cell types in the overall MMP inflammatory response. Knowing more about the MMP response of the immune system can provide us with a better understanding of regulation of MMP expression. For example, MMP9 cleaves and activates interleukin-8 (IL8), a cytokine that enhances neutrophil recruitment to the site of infection . MMP9 haSimilarly, dendritic cells produced more collagenase MMP1 than other cell types, but this response was significantly attenuated when all three cell types were cultured together b. MMP1 iin vitro culture systems are limited by the inability to represent all of the immune mediators found in the host environment. In particular, a more complex multi-dimensional system that can more closely mimic the in vivo inflammatory response is required to generate a comprehensive understanding of the process underlying periodontal disease [Co-culture models differ from other systems because of their unique ability to focus on the cellular communication between different cell types over time. Standard  disease . There a disease ,21,22,23in vivo. With this in mind, we chose to use equal concentrations of each cell type because it is easily repeatable and can be plated in the transwell plates at a reasonable density.The three-cell transwell co-culture system developed for this study is not without its own advantages and disadvantages. We used a heterotypic, transwell co-culture system that does not allow contact among the different cell types by separation using membranes as barriers between the cells. By removing cell–cell physical interactions, we can focus on the effects of cell signals elicited by the agonist or from other cells in the co-culture. Preventing cell-to-cell contact allows us to use cells from different individuals that would otherwise be incompatible. One limitation of this study is that the cells are not necessarily in the same ratios as they would be In conclusion, a number of factors could be causing the attenuation of dendritic cell expression of MMP9 and MMP1 in the three-cell co-culture. This study provided insight into MMP regulation in the host environment and the effects of a proinflammatory agonist, HagB. Learning more about these signaling pathways and MMP involvement may lead to identification of possible targets for therapeutic approaches. Further analysis will look for connections between the MMP responses over time and the associated cytokine responses, which will provide more information on the inflammatory response. In silico models using bioinformatics are being developed to help us study and analyze inflammatory responses in environments like the periodontal tissues where multiple factors are involved . PreviouP. gingivalis (Pg strain 381) was cloned and His-tagged into the vector pQE31 and expressed in Escherichia coli , which was obtained and prepared similarly to what was previously described [E. coli was grown at 37 °C in selective Luria-base (LB) broth with ampicillin and kanamycin A. After 4 h, 1 mM Isopropyl B-D-1-thiogalactopyranoside (IPTG) per liter of culture was added to induce HagB expression. After an additional 4 h of incubation, 10.0 mL of 1.54 M sodium azide (NaN3) per liter of culture was added to arrest growth. The culture was pelleted and washed in 10.0 mL of 140 mM phosphate buffered saline (PBS). The PBS was removed, and the cells were resuspended in 10 mL of Buffer A  to lyse the E. coli. The cells were pelleted and the supernatant containing the HagB was collected.Hemagglutinin B (HagB) from escribed . Briefly2PO4, 0.01 M Tris acid, pH 8.0, followed by a second wash with 8.0 M Urea, 0.1 M NaH2PO4, 0.01 M Tris acid, pH 6.3. To refold the HagB, a buffer containing 6.0 M Urea, 0.5 M NaCl, 0.01 M Tris acid, 20.0% glycerol, pH 7.4 was applied to the column. The next refolding buffer applied was the same buffer but with 3.0 M Urea followed by buffer without Urea. The last buffer  added to the column was a refolding buffer with imidazole to elute the HagB using a fraction collector. The protein concentration of each fraction was obtained using a NanoDrop , and fractions containing the most protein were pooled.To purify the HagB, affinity chromatography was performed using the BioRad Biologic low-pressure pump and a column containing Profinity IMAC Ni-charged resin , which binds His-tagged proteins. After the column was equilibrated with Buffer A, the supernatant containing HagB was passed through the column. This elution was collected and discarded since the proteins remained in the column. The affinity-attached HagB was washed with 8.0 M Urea, 0.1 M NaHThis protein sample was applied to a desalting column  to remove imidazole and for further purification using the BioRad Biologic low-pressure pump. The column was equilibrated with a refolding buffer of 0.5 M NaCl, 0.01 M Tris acid, 20.0% glycerol, pH 7.4. The pooled fraction sample was run through the column followed by the refolding buffer without urea. The protein concentration was quantified using a NanoDrop, and fractions containing the most protein were pooled together.P. gingivalis. To determine the protein concentration of the sample more precisely, a BCA protein concentration assay was utilized, and found to be approximately 0.445 mg/mL (Pierce bicinchoninic acid (BCA) Protein Assay Kit, ThermoFisher Scientific, Rockford, IL, USA). The final concentration of LPS was 2.44 ng LPS/1.0 µg HagB . An SDS-Page of the sample was performed to verify purification of HagB using a NuPAGE Novex 4% to 12% Bis-Tris protein gel. This produced a solid band at about 46 kDa. A MALDI-TOF mass spectrometry analysis confirmed the presence of HagB in this band with the greatest number of matches and sequences of all proteins found. HagB was the only protein found from g) at 4 °C for 10 min, and then resuspended in LGM-3 to 1 × 105 viable cells/mL. CD4+ T-cells  were activated using a human T-cell Activation/Expansion kit  in TexMACS medium (Miltenyi Biotec) for 3 days at 37 °C with 5% CO2. The T-cells were then collected, pooled, and a viable cell count was obtained. The T-cells were resuspended to 1 × 105 viable activated cells/mL in LGM-3. Primary first passage GE keratinocytes were used, which were previously prepared and stored in liquid nitrogen [Dendritic cells, GE keratinocytes, and T-cells were chosen for this experiment due to their presence in the periodontium and their innate immune functions. Human monocyte-derived immature myeloid dendritic cells  were thawed, washed with LGM-3 , counted, pelleted by centrifugation  and inserts from the transwell 24-well polystyrene plates . 200 µL of 1 × 10h 5% CO2 a. This cTo compare the responses between different cell types, single-cell responses, and the response of all three cell types together, combinations of cell types and single cells were set up in the same manner b. In totg to pellet any cells or debris, and supernatants were stored at −80 °C. The plates were placed on the shaker and incubated at 37 °C, 5% CO2 for 64 h. Samples were collected in this same manner at 2, 4, 8, 16, and 32 h. At 64 h, 2.0 mL centrifugal filter tubes were used to pool the media, one tier at a time. HagB was then added at a concentration of 10.0 µg/mL. Preliminary tests were used to determine the appropriate concentration of HagB to use by testing varying concentrations and their respective dendritic cell response. An equal amount of buffer was added to control wells. Plates were placed on a titer plate shaker at a low speed for 60 s to diffuse the treatment. 200 µL of supernatant was collected from each well as a time zero sample, and 200 µL of LGM-3 was added back to each well. Samples were centrifuged for five minutes at 0.8× 100  (n = 3) (n = 9) using multiplex immunoassays  read on the Luminex100  read on the Luminex (n = 3) a. After minex100 b. These 100 and readout files . First, the 0 to 32 h results from the immunoassay  were analyzed (n = 3). The responses of the control plate (HagB diluent) were subtracted from the responses of the test plate (HagB) to assess only the effect of the presence of HagB. A two-way fixed effect ANOVA was fit to log-transformed concentrations of the MMPs . Pairwise group comparisons were conducted using the post-hoc Tukey’s honest significance test. A significance level of 0.05 was used determine statistically significant differences between groups.MMP concentrations were interpolated from curves constructed from the MMP standards and their respective median fluorescence intensity (MFI) readings on the Luminexn = 9). Responses from the control plate (HagB diluent) were subtracted from the responses from the test plate (HagB) to assess only the effect of HagB. Once again, results were analyzed using a two-way fixed effect ANOVA fit to log-transformed data and pairwise group comparisons were made.The co-culture experiment was then repeated three times, and three repeats from each experiment at 64 h were included in the immunoassay (


The excellent purity of the monomers combined with the high vacuum techniques resulted in controlled polymerization with high molecular and compositional homogeneity. FT-IR, as well as circular dichroism, were employed to investigate the secondary structure of the polymers, while DLS, SLS and ζ-potential were utilized to study the aggregates formed in aqueous solutions, as well as their pH responsiveness. The results revealed that the randomly distributed monomeric units of glycine or alanine significantly influence L-histidine’s structure. Depending on the pH, aggregates with a different structure, different molecular characteristics and a different surface charge are formed, potentially leading to very interesting bioapplications.The highly diverse and sophisticated action of proteins results from their equally diverse primary structure, which along with the nature of interactions between the amino acids, defines the higher self-assembly of proteins. The interactions between amino acids can be very complicated, and their understanding is necessary in order to elucidate the protein structure-properties relationship. A series of well-defined hybrid-polypeptidic diblock copolymers of the type  Synthetic polypeptides synthesized via controlled polymerization exhibit complex compositions and architectures and have been investigated over recent decades as a promising system for many bio-applications ,2,3. TheN-carboxyanhydrides (ΝCAs) [In order to exploit polypeptide characteristics, the predetermination of their properties by adopting the appropriate macromolecular architecture, molecular weights and composition are necessary, and in order to make these systems functional, control of their molecular characteristics is of key importance. The most commonly used method for the synthesis of block copolypeptides is the ring opening polymerization (ROP) of s (ΝCAs) . The ROPs (ΝCAs) ,14. In as (ΝCAs) . The PEGs (ΝCAs) . Hydroges (ΝCAs) ,18. Furts (ΝCAs) .Among the polypeptides that have been used for block polymerization with PEG, Poly(L-histidine) (PHis) has unique characteristics which render it a suitable material for biological applications. The amphoteric nature of PHis, due to its unsaturated nitrogen at its imidazole ring, can turn it from soluble at pH 6.3 to insoluble at pH 7.4 , thus reb-poly polymers that formed micelles in aqueous solutions. They reported that the copolymer pKa value can be controlled by tuning the histidine to phenylalanine molar ratio in the copolypeptide block [b-polyglycine to study their supramolecular assembly and found that poly(glycine) offers flexibility and compliance to the formulated vesicles. Papadopoulos et al. [γ-benzyl-L-glutamate)-b-polyglycine (PBLG-b-PGly) block copolypeptides. They found that due to the multiple chain folding of PGly, the lamellar structure of PBLG was stabilized. Finally, Yu et al. [The random distribution of monomeric units of hydrophobic peptides along the PHis chain can alter its secondary structure and its degree of protonation resulting in different solubilities and aggregation properties for the formation of nanoparticles. These possible variations can be exploited through the design of systems with controllable secondary structures and self-assembly ability. The type and the ratio of the randomly distributed hydrophobic units within PHis are expected to lead to systems with different self-assembly properties . Kim et de block . Two othde block  have syns et al.  also stuu et al.  used polα-helix and β-sheet [β-sheet conformation was high for the l-alanine polymers while α-helix was high for the polymers with the d-isomer [Poly has been widely used to form hydrogels that present reverse thermal gelation properties ,31. In t β-sheet ,35 second-isomer . Additiod-isomer . m-PEO-b-poly(His-co-Gly) and m-PEO-b-poly(His-co-Ala) with hydrophobic peptide (Gly or Ala) to histidine peptide molar ratios of 10%, 20%, and 40% is presented  was supplied from Fisher Scientific  (99.91%), special grade for peptide synthesis with less than 50 ppm active impurities and was further purified by short-path fractional distillation under high vacuum. The middle fraction was always used. Trifluoroacetic acid  was also purchased from Fischer Scientific. Thionyl chloride (SOCl2)  was obtained from Acros Organics. Triethylsilane (99%), Limonene (97%) and diethyl ether (≥99%) were purchased from Aldrich. Purification of benzene , dichloromethane  and tetrahydrofuran ) was performed using standard high vacuum techniques reported elsewhere [n-BuLi. Distilled water was further purified by a Milli-Q water purification system . End-functionalized amine-terminated methoxy protected poly(ethylene oxide) (m-PEO-NH2) having molecular weights of 9.95 × 103 g/mol were purchased from Aldrich and were employed as monofunctional macroinitiators.Boc-His(Trt)-OH (>99%) was purchased from Christof Senn Laboratories AG . Triphosgene (99%) was acquired from Acros Organics . Triethylamine  was dried over calcium hydride for one day and then distilled and stored over sodium. Then, the desired amount was distilled to other apparatus prior to use. lsewhere . AcetoniThe synthesis of Trt-His-NCA was accomplished in two steps and has been reported elsewhere in detail . Brieflyα-amino acid , which was dissolved in acetonitrile. Limonene (6 mL) and triphosgene 7,32 g (24.6 mmol) were added at 70 °C under an inert atmosphere. After filtration, the filtrate was recrystallized by hot filtration. Finally, the NCA was dried under vacuum overnight .The NCA of glycine (Gly-NCA) was synthesized from the corresponding l-alanine 5.0 g (56.1 mmol) was suspended in acetonitrile and triphosgene 6.16 g (20.7 mmol) was added at 70 °C. After filtration, Ala-NCA was then dissolved and dried several times with ethyl acetate under high vacuum, in order to remove the excess triphosgene. Finally, Ala-NCA was recrystallized in a system of ethyl acetate and hexane three times under high vacuum. The purified NCA  was stored under an inert atmosphere at 0 °C.The synthesis of the alanine NCA (Ala-NCA) was carried out following to a previously published method . l-alaniAll manipulations and polymer synthetic procedures were performed under high vacuum in custom-made glass reactors, equipped with break-seals, high vacuum stopcock, glass-covered magnets and constrictions for the addition of reagents under the guidelines of the high vacuum techniques . The ream-PEO-NH2 with molecular weight 9.95 × 103 g/mol (0.02 mmol) were added into the apparatus. This amount of the macroinitiator  was kept constant for each of the six synthesized polymers. The custom-made glass reactor was connected again to the vacuum line, it was evacuated and the apparatus was remained connected overnight. The next day, 20 mL of dry benzene were distilled and the macroinitiator was dissolved. The solution was stirred for two hours and benzene was distilled off to dryness. DMF (20 mL) was distilled followed by dissolution of the macroinitiator. Subsequently, the apparatus was inserted again in the glove box for monomer addition. The apparatus was equipped with a side ampoule with a ground joint and a constriction. The monomer amounts were added in order to obtain glycine or alanine to histidine molar ratios at 10%, 20%, and 40%. Trt-His-NCA  and Gly-NCA  were added for the polymer with 10% glycine to histidine molar ratio, while 0.01 g of Ala-NCA (0.088 mmol) (instead of Gly-NCA) was added to the polymer with a 10% molar ratio of alanine (instead of glycine) to histidine. The corresponding amounts for the polymers with 20% of glycine to histidine molar ratio were: 0.330 g (0.78 mmol) of Trt-His-NCA and 0.0188 g (0.19 mmol) of Gly-NCA, while 0.0214 g (0.19 mmol) of Ala-NCA (instead of Gly-NCA) was added to the polymer with a 20% alanine (instead of glycine) to histidine molar ratio. For the polymer with 40% glycine to histidine molar ratio, 0.310 gr (0.73 mmol) of Trt-His-NCA and 0.045 gr (0.45 mmol) of Gly-NCA were added, while 0.051 g (0.45 mmol) of Ala-NCA (instead of Gly-NCA) were added to the polymer with the 40% of alanine (instead of glycine) to histidine molar ratio.A custom-made glass reactor was initially attached to the vacuum line through the ground joint and was evacuated and flame dried several times. It was subsequently inserted into the glove box and 0.2 g The ground joint of the side ampoule of the apparatus containing the NCAs was subsequently connected to the vacuum line and, after evacuation, 5.0 mL of dry DMF were distilled. The reactor was removed from the vacuum line through heat sealing a constriction and the mixture of NCAs was dissolved. The rupture of the break-seal of the ampoule followed resulting in addition of the solution of the NCAs to the solution of the macroinitiator. After termination of the polymerization, the polymer was precipitated in diethyl ether and dried under high vacuum.2Cl2 (10 mL) and an equal volume of trifluoroacetic acid (TFA) was added. The polymer was completely dissolved and was left to be deprotected for 1 h at room temperature. An equimolar amount (with respect to the number of histidine monomeric units) of triethyl silane was added. The solution was distilled in the vacuum line in order to remove all solvents. The solid that remained was dissolved in water and was dialyzed against 2 liters of Milli-Q water with pH~3 (adjusted with a dilute aqueous solution of HCl) twice, twice in Milli-Q water with pH~9 (adjusted with a dilute aqueous solution of NaOH) and twice in pure Milli-Q water. Finally, the aqueous polymer solution was freeze dried to result in the corresponding polymer. The obtained polymer was 0.15 g for each polymerization.The polymer was suspended in CHSize-exclusion chromatography. Size-exclusion chromatography (SEC)  was used to determine the Mn and Mw/Mn values. It was composed of a Waters Breeze instrument equipped with a 2410 differential refractometer and a Precision PD 2020 two angle  light scattering detector. A 0.10% TFA (v/v) solution of water/acetonitrile  at a flow rate of 0.8 mL/min at 35 °C was used as the carrier solvent. Three linear Waters hydrogel columns were employed. NMR spectroscopy. Proton nuclear magnetic resonance (1H NMR) spectroscopy (300 MHz) was performed using a Varian Unity Plus 300/54 spectrometer  in deuterated dimethylformamide (DMSO).FT-IR. Fourier transform infrared spectroscopy (FT-IR) measurements were performed with a Perkin Elmer Spectrum One instrument , in KBr pellets at room temperature, in the 450–4000 cm−1 range. Circular Dichroism. Circular Dichroism was performed with a Jasco J-815 model, featuring a Peltier model PTC-423S/15 thermo stabilizing system . A 1 mm Quartz Suprasil cell was employed. Typical concentrations were about 7 × 10−4 g/ml.Dynamic and static light scattering. Dynamic light scattering measurements were carried out with a Series 4700 Malvern system  composed of a PCS5101 goniometer with a PCS7 stepper motor controller and a Cyonics variable power Ar+ laser, operating at 690 nm and with 10 mW power, a PCS8 temperature control unit, and a RR98 pump/filtering unit. Correlation function analysis included the cumulant method and the Contin software . The correlation function was collected at 90°. Measurements were carried out five times for each sample and were averaged. Static light scattering measurements are taken with the same DLS instrument in a 30°–150°angular range, measured every 10 degrees. Measurements were carried out three times for each sample and were averaged. Toluene was used as the calibration standard. The dn/dc values were calculated from literature data [ure data . The anaZeta potential. The zeta potential of the synthesized polymers was measured by the technique of microelectrophoresis, using a Malvern Zetasizer 3000HSA instrument  at room temperature at 633 nm. All the measurements were the average of at least three trials. The ζ-potential was calculated from the corresponding electrophoretic mobilities, μE, using the Henry’s correction of the Smoluchowski equation. Sample preparation. All measurements were carried out in isotonic Tris buffer (10 mM) with NaCl (150 mM) at pH = 7.4, in isotonic 2-(N-morpholino)ethanesulfonic acid (MES) buffer (10 mM) with NaCl (150 mM) at pH = 6.5, and in isotonic MES buffer (10 mM) with NaCl (150 mM) at pH = 5.0. Millipore water was thoroughly filtered with 0.1 μm filters and used directly for the preparation of the solutions. Initially, the polymers were dissolved in THF, followed by a dropwise addition of the appropriate amount of buffer solution, and removal of THF through heating and weighting the remained solution. In some cases, we added pure Milli-Q water in case that the weight was lower than the appropriate one. Typical concentrations were 5.0 mg of each polymer at 5.0 mL of the respective buffer. Sonication was performed when needed. No NaCl was used for the preparation of the samples measured with Zeta potential.m-PEO-b-poly(His-co-Gly) and m-PEO-b-poly(His-co-Ala) are depicted in 3 g/mol. The high purity of the synthesized NCAs is crucial for maintaining the living nature of the polymerization as well the complete monomer consumption. If traces of protic impurities remain in NCAs, the polymerization will slow down or will be completely terminated, due to termination of the living active ends. In every polymerization, the macroinitiator (m-PEO-NH2) was dissolved in purified, dried benzene and then the benzene was distilled off to dryness. This step was necessary in order to remove residual water in PEO, which can trigger NCA polymerization. The CO2 produced from the polymerization was also periodically removed by degassing under high vacuum. All the copolymerizations showed a lack of heterogeneity during the polymerization in contrast to the homopolymerization of histidine NCA for the synthesis of m-PEO-b-poly(His) . Additionally, no indication for the Trt protecting group was found by 1H NMR of the final polymers. The 1H NMR spectrum is provided in b-P(His-co-Gly) and PEO-b-P(His-co-Ala) are shown in The synthetic routes followed for the preparation of the hybrid-copolypeptides ous work ), which ous work . This is750 cm−1 . The pea−1. This vibration is assigned to random coil conformation. The adoption of the random coil motifs by these polypeptides in the solid state renders the formation of an intramolecular network difficult. The polypeptide with the glycine residues at 40% molar ratio displays a peak at 1624 cm−1, which suggests the β-sheet formation. It is well-known that Poly(Gly), depending on the casting conditions, can very often exist in β-sheet structures, which influence the segmental mobility [−1, corresponding to amide I band and one at 1548 cm−1, corresponding to amide II band. Both vibrations are attributed to α-helix formation. Gitsas et al. suggested that although pure Poly(Ala) stabilizes both α-helices and β-sheets, α-helices are thermodynamically more stable in the copolypeptides [The conformational analysis of each diblock polypeptide was performed using FT-IR in solid state after lyophilization and CD in the solution state in Milli-Q water. All the synthesized polypeptides exhibit an intense amide I band, which is governed mainly by the stretching vibrations of the C=O group. In the dry state, the polypeptides with the glycine residues at 10% and 20% molar ratios, as well as the polypeptides with alanine residues at 10% and 20% molar ratios, display an intense amide I band around 1640 cmmobility . The polpeptides . The cham-PEO-b-poly(His-co-Gly). Only PHis contributes and governs the secondary structures. All of the glycine containing diblocks exhibited a broad negative trough below 200 nm and a positive peak above 220 nm. These results are in agreement with the random coil conformation, which is adopted for all the diblocks with the different molar ratios of Gly to His residue. The same motif is maintained at every pH. It can also be observed that the θ magnitude for the diblock with 40% molar ratio of glycine residue to histidine residue decreased due to the reduced monomeric units of histidine. The same conformation was obtained for all the polymers including alanine moieties, despite the fact that alanine is not achiral. Specifically, all the polypeptides of the type PEO-b-poly(His-co-Ala) provide a broad negative peak below 200 nm and a positive one above 220 nm, which can be assigned to the random coil conformation. This conformation is also maintained at every pH. All the synthesized polymers were examined with CD as a function of temperature showing no change in their secondary structure.Circular dichroism spectroscopy was used to reveal the secondary structure of these polypeptides and particularly to elucidate how the randomly distributed residues of glycine and alanine influence the conformation of PHis in water . CD measm-PEO-b-poly(His), the conformation is almost identical at pH 5.0, as the random coil conformation is also preferred by the polymer. The protonated moieties of histidine undergo repulsion, which is a deterrent factor for the formation of an ordered structure. As the pH value increased the diblock m-PEO-b-poly(His) presented a different secondary structure as β-sheet conformation developed. The synthesized polymers of the current work do not seem to follow this trend, probably because the hydrophobic monomeric units of glycine or alanine do not allow the histidine monomeric units to form organized structures. The choice of an appropriate hydrophobic amino acid to be randomly distributed among the PHis chain is crucial for the control of the properties of these systems. In the same work [b-poly(His-co-Leu) to α-helix at 40% molar ratio; however, this did not happen in this work in the case glycine or alanine monomeric units.These results are comparable with CD spectroscopy studies formerly carried out by our group on similar systems . In the ame work , it was RH) values to those observed at the same temperature at three different pH values  for each different sample. The results from DLS are illustrated in Ka of the samples, which plays a crucial role for the aggregation properties. For the diblocks m-PEO-b-poly(His-co-Gly), the increase of the molar ratio of the glycine to histidine residue has, as a result, an increase of the RH at every pH. Additionally, as the pH value decreases for all the diblocks containing glycine monomeric units, the KCounts of the scattering light are also lower, suggesting partial disruption of the aggregates. For the diblocks m-PEO-b-poly(His-co-Ala), the increase of the alanine to histidine molar ratio follows no particular trend. Furthermore, all the alanine containing aggregates are swollen upon lowering the pH. The reduction of the KCounts of the scattering light is also observed with the decrease in pH for all the samples containing alanine monomeric units. The large size of polymeric structures (over 100 nm) is most likely due to the formation of secondary aggregates that are clusters of single aggregates. The participation of the hydrophobic monomeric units of glycine and alanine into the PHis chain increases the dimensions of the structures compared to the structures of m-PEO-b-poly(His) [RG) to RH (RG/RH ratio) depends on the morphology of the aggregates formed. RG/RH values close to 1 can be attributed to the formation of vesicles, whereas smaller values are generally attributed to spherical micelles  [RG/RH ratio also decreases. Note that these RG/RH ratio values are lower than the value known for a hard sphere (RG/RH = 0.78). At lower pH values, the dimensions of the aggregates are much higher than 100 nm, and the obtained RG values are apparent.DLS was utilized to elucidate the aggregation behavior of the synthesized polymers. PHis has the ability to become hydrophilic and interact with water at lower pH due to its protonation . The paroly(His) . Static ly 0.78) . All theRG/RH obtained close to 1, while the polymers with 10% and 20% probably form core-shell micelles. The dissolution of the synthesized polymers in aqueous media, directs the PEO blocks towards the aqueous phase, while the hydrophobic polypeptidic domain of the diblocks is physically entangled in the inner matrix. The formed hydrophilic PEO shell presents no surface charges, which offers “stealth” properties to the nanostructures against the reticuloendothelial system. As a result, they become invulnerable to macrophages. The results of zeta potential measurements are presented in α-amino acid NCAs, we successfully synthesized a series of novel diblock polypeptides of the type m-PEO-b-poly(His-co-Gly) with glycine to histidine molar ratios at 10%, 20%, 40% and of the type m-PEO-b-poly(His-co-Ala) with alanine to histidine molar ratios at 10%, 20%, 40%. The molecular characterization results for all synthesized terpolymers show that the polymers had a high degree of molecular and compositional homogeneity. The study of their secondary structure in solid state revealed that the polypeptide with 40% molar ratios of glycine to histidine adopts β-sheet formation, while the polypeptide with 40% molar ratios of alanine to histidine adopts α-helix formation. All other synthesized polymers adopt random coil formation. The random coil conformation was revealed for all the synthesized polymers in water. Studies on the self-assembly of these terpolymers in aqueous solutions showed that they form aggregates with the general trend of the dimensions to increase as the pH decreases. All the samples at pH 7.4 showed a tentative micellar morphology, with the exception of the 40% Gly-containing polymer that probably exhibits a vesicular structure. Zeta potential experiments revealed that the surface charges of the nanostructures of all the synthesized polymers at pH 5.0 are larger and positive in comparison with the surface charges of the nanostructures at pH 7.4 and pH 6.5, which is almost zero. This shows that at pH = 7.4 and 6.5 the PEO remain in the outer periphery, while at pH = 5.0 the strong protonation of PHis results in more swollen chains that reach the shell of the nanoparticle. The study of the properties of the synthesized diblocks revealed that based on the composition and the nature of the hydrophobic amino acid along the PHis chain, it is possible not only to alter the aggregation morphology of the nanoconstructs but also their pH-responsiveness. It was shown that at lower molar ratios of the hydrophobic amino acids Gly and Ala along the PHis chain, PHis becomes very hydrophobic and a significant decrease in pH is required to disrupt the aggregates, as compared to the pKa of 6.5 of pure PHis. This could be an interesting property for the encapsulation of hydrophobic drugs within the aggregate core to be released only under lower pH conditions, and therefore remaining stable during circulation within the blood compartment to be delivered only upon internalization within the cells.Employing high vacuum techniques and the ROP of


P=0.033 and P=0.063). And it was demonstrated that PMRT were independently associated with increased OS according to univariate and multivariate analyses. However, no significant differences in BCSS or OS were observed between the groups stratified by the number of positive lymph nodes. In conclusion, PMRT significantly improved OS for TNBC patients with T1-2 and 1-3 lymph nodes involved. Additional prospective studies are needed to provide a stronger evidence base for choosing patients for PMRT.There is consensus on the routine use of postmastectomy radiotherapy (PMRT) in patients with four or more positive axillary lymph nodes. However, the benefits of PMRT in patients with T1-2 and 1-3 involved lymph nodes still remain controversial. Data from the Surveillance, Epidemiology, and End Results Program (SEER) of the United States between 2010 and 2012 were used to analyze the outcomes of 675 triple-negative breast cancer (TNBC) patients with T1-2 and 1-3 lymph nodes involved. Those patients were subdivided into radiotherapy (RT) (312) and no-RT groups (363). After a median follow-up time of 37 months, Kaplan-Meier analysis showed that PMRT significantly improved overall survival (OS) but not breast cancer-specific survival (BCSS) in the total cohort of 675 patients ( Breast cancer is a highly heterogeneous disease, which is reflected in different clinical, pathologic, and molecular features. Currently, breast cancers are classified into four molecular subtypes including, luminal A, luminal B, human epidermal growth factor receptor 2 (HER2) overexpressed, and triple-negative breast cancer (TNBC) subtypes according to estrogen receptor (ER), progesterone receptor (PR), and HER2 status by gene expression profiling , 2. The Adjuvant radiotherapy plays a pivotal role in the treatment of breast cancer, since it can reduce the risk of loco-regional recurrence and overall mortality , 6. And Given the poor prognosis and aggressive characteristics of TNBC patients and the controversy surrounding PMRT as appropriate for patients with small tumors with 1–3 positive nodes, our study is designed to investigate the effect of postmastectomy radiotherapy on OS and breast cancer-specific survival (BCSS) among TNBC patients with T1-2 and 1-3 positive lymph nodes by utilizing population-based SEER data, and to further determine which patients are most likely to benefit from PMRT.A summary of demographic and clinical characteristics is shown in P=0.033 and P=0.063). In addition, no significant differences in OS was observed between the groups stratified by the number of positive lymph nodes; P=0.073, P=0.455, P=0.144 for 1 LN, 2 LNs, and 3 LNs. there was no significant difference in BCSS when patients were stratified by the number of LN; P=0.181, P=0.452, and P=0.176 for 1 LN, 2 LNs and 3 LNs.We used the Kaplan-Meier plot and log-rank tests to compare BCSS and OS between different subgroups according to lymph node their status and the results are illustrated in P=0.009 for OS and HR, 1.484 with a 95%CI, 1.035 to 2.127 and P=0.032 for BCSS. Compared with tumors sized 2 cm to 5 cm (T2), patients with ≤2 cm (T1) tumors had a better OS and BCSS with univariate analyses; HR, 0.617 with a 95%CI, 0.416 to 0.917 and P=0.017 for OS and HR, 0.548 with a 95%CI, 0.353 to 0.852 and P=0.008 for BCSS. This result retained significance in multivariate analyses of OS and BCSS  and 20-year rates of breast cancer mortality . But theIn clinical practice, considering the delayed complications from radiation including cardiac toxicity, lymphedema, skin fibrosis, and so on, some surgeons and radiation oncologist do not recommend PMRT to patients with 1-3 positive axillary lymph nodes. But TNBC is a special subtype of breast cancer. Patients with TNBC are likely to suffer poorer survival and are prone to early locoregional recurrence and distant metastasis. Previous studies have demonstrated that different molecular subtypes had different response to PMRT. Wen et al.  found thAlthough no statistical difference was observed for BCSS among the total cohort and by stratified analysis, a benefit trend was reflected from Kaplan-Meier analysis which was consistent with the trend in OS, thus these observations should be interpreted with caution. We speculated that the small percentage of patients who have died from breast cancer and the small sample size of patients who died in subgroup analysis could be one potential explanation for the finite benefits from PMRT. And hopefully will extending the follow-up time make our results more satisfactory.There are several limitations of our study. Firstly, we were compelled to focus on short follow-up time because the information regarding HER-2 expression in the SEER database was not available until 2010. But for the TNBC subtype, an early peak of recurrence occurs within the first 2-3 years after diagnosis. Secondly, the SEER database is absent from information about LRR. However, information about chemotherapy is offered by the latest version of the SEER database. And we endeavored to mitigate several confounding variables, such as endocrine and trastuzumab treatment by selecting TNBC as the object of our study rather than other subtypes.In conclusion, our study shares some similar results with the recent literature that there is a significant improvement on OS after PMRT among TNBC patients with T1-2 and 1-3 positive axillary lymph nodes. In accordance with current consensuses and guidelines, we suggest that PMRT should be strongly considered in T1-2N1 breast cancer patients particularly in TNBC, an aggressive and poor prognosis disease. Additional prospective randomized studies are needed to provide a stronger evidence base for selecting patients for PMRT.The data in the SEER database do not require informed patient consent because cancer is a disease reported by every state of the United States. But a Data-Use Agreement for the SEER 1973–2014 Research Data File has been completed.in situ disease, and no record of radiotherapy. We calculated follow-up durations from January 1, 2010 to December 31, 2014 to ensure adequate follow-up duration.SEER*Stat version 8.3.4 was used to generate a case listing. We identified 675 patients screened according to the following inclusion criteria: female; year of diagnosis from 2010 to 2012; age of diagnosis between 20 years and 80 years; breast cancer as the first and only malignant cancer diagnosis; pathologically confirmed invasive ductal carcinoma-not otherwise specified (ICD-O-3 8500/3), unilateral cancer; TNBC subtype ; histological grades I-III; AJCC T stagesI-II; known tumor size category; known lymph node status; record of chemotherapy therapy, mastectomy received. We excluded patients with inflammatory breast cancer, P-values were two-sided and P<0.05 were considered statistically significant. All statistical analyses were carried out using SPSS version 22.0 software .Patients were divided into RT group and no-RT group categorized by radiotherapy status. A Chi-squared test was used to compare the demographic and clinical characteristics of the included cases between the two groups. The Kaplan-Meier analysis was performed to generate survival curves, and the log-rank test was used to compare the unadjusted BCSS and OS rates of patients with different lymph node status. BCSS was measured from the date of diagnosis to the date of breast cancer death. OS was defined as the time from the date of diagnosis to the date of death due to any cause (including breast cancer) or the last follow-up. Adjusted hazard ratios (HRs) with 95% confidence interval (CI) were calculated using a Cox proportional hazard regression model to estimate the outcome-related factors. All


The microenvironment is an important factor of stem cells regulating their maintenance, survival, and differentiation. The glycation of proteins with reducing sugars through nonenzymatic reactions induces the collagen cross-linking, which causes tissue stiffening, which is enhanced during aging and diabetes. In this study, we aimed to analyze the effect of glycated collagen on the stem cell culture and differentiation. The collagen type 1 was modified by glycation with mannose, rhamnose, arabinose, and glucose. After the culture of mesenchymal stem cells on the coated surfaces with glycated collagen, the differences in cell adhesion, proliferation, and differentiation were compared. The results showed that the modifications did not induce apoptosis or cause cell death. However, the culture of cells on modified collagens improved the proliferation. It was found that the mannose-modified collagen stimulated the adipogenic differentiation of stem cells, and rhamnose-modified collagen supports the differentiation into both osteogenic and insulin-producing cells. The low concentration of monosaccharides during glycation process improved the characteristics of the matrix protein in favor of stem cell differentiation. Modification of the collagen by glycation might be used as a tool to improve natural polymers for material-induced stem cell differentiation in the future. Stem cell differentiation was directed not only by soluble biofactors but also by other factors in the microenvironment of stem cells. The physical aspects, like surface topography , stiffneIn certain circumstances, proteins can also undergo spontaneous modifications in vivo and contribute to age-related diseases. Under the hyperglycolytic conditions, for example, the proteins experience nonenzymatic posttranslational modification leading the formation of advanced glycation end-products (AGEs). Type 1 diabetic patients are especially susceptible to AGE formation. The oxidative condition caused by the accumulation of AGEs in the tissue might lead to biophysical disorders, like Alzheimer, cardiovascular diseases, diabetes, and renal failure . The AGEIn this study, collagen type 1 was modified by glycation. The effect of this nonenzymatic alteration with four monosaccharides (glucose (G), mannose (M), arabinose (A), and rhamnose (R)) on the cell morphology and the direction of the differentiation was analyzed. The primary aim was to demonstrate the biological effects of the modified collagen by glycation with various monosaccharides on stem cell response and differentiation.2, humidified atmosphere. The medium was refreshed every two days. The cells were expanded in conventional plastic culture flasks . Unless it was mentioned, the cells were seeded on the glass surface for the assays at the density of 3000 cells/cm2.Pancreatic islet-derived mesenchymal stem cells (PI-MSCs) were isolated from rat pancreatic islets by explant and characterized, in the previous study . The celD-(+)-glucose monohydrate, D-(+)-mannose, D-(−)-arabinose, and L-rhamnose monohydrate were supplied from Sigma-Aldrich . 100 mM monosaccharide solution was prepared in phosphate-buffered saline (PBS) buffer  separately and mixed with human collagen type I  to 1 mg/ml final solution in PBS buffer. Protein-monosaccharide mixtures were incubated for 14 days at 37°C. Then, collagen solution was dialyzed in Slide-A-Lyzer MINI Dialysis Device  for 16 h against 1000 times the volume of sample with PBS at 4°C.μg/cm2 for 24 h at 37°C in 5% CO2 humidified atmosphere. The residue solution was removed, and the surfaces were briefly washed with PBS buffer. Without drying the surfaces, cells were cultured. The group of surface coating with unmodified collagen was named “Col,” and the groups coated with glucose-, mannose-, arabinose-, and rhamnose-modified collagen were referred as “Col-G,” “Col-M,” “Col-A,” and “Col-R,” respectively.Glass surfaces were coated with collagen at the concentration of 10 For staining of the actin cytoskeleton with phalloidin-FITC (Sigma-Aldrich), the cells were washed in PBS briefly, fixed with paraformaldehyde (2% in PBS), permeabilized in Triton-X100 (0.1% in PBS) for 60 s and incubated in the phalloidin-FITC solution  for 20 min. Following washing twice in PBS, the samples were mounted with mounting medium containing 4′,6-diamidino-2-phenylindole . The cells were investigated under the fluorescent microscope .After culture, the medium was replaced with RPMI 1640 basal medium supplemented with 10% WST-1 reagent . After incubating for 2 h, the absorbance at 450 nm was measured by the spectrophotometer , according to the instruction of the manufacturer.Cell adhesion was evaluated after 16 h of incubation. Attached cells were counted by WST-1 solution . As a control, cells cultured on the uncoated glass surface were used.Cells were fixed in methanol  at 4°C for 10 min. After permeabilization with 0.025% Triton X-100 (Merck), the samples were incubated with 1.5% blocking serum (Santa Cruz Biotechnology) in PBS for 30 min at 37°C. After washing three times with PBS, samples were incubated overnight at 4°C with the primary antibody (Supplementary Cytotoxicity was determined by lactate dehydrogenase (LDH) assay with Cytotoxicity Detection Kit LDH (Roche), according to the manufacturer's instructions. The cell culture on the uncoated glass surface was used as a control.Total RNA was extracted with High Pure RNA Isolation Kit (Roche), according to the manufacturer's instructions. After synthesis of single strand cDNA by Transcriptor High Fidelity cDNA Synthesis Kit (Roche), gene expressions were carried out by LC480 DNA SYBR Green I Master (Roche) with gene-specific primers (Supplementary μM dexamethasone (Sigma-Aldrich), 0.05 mM ascorbate-2-phosphate (Sigma-Aldrich), 10 mM β-glycerophosphate (Sigma-Aldrich), 1% penicillin/streptomycin (Gibco), and 10% FBS. The medium was refreshed twice a week. At the end, the differentiation was estimated by Alizarin Red staining. For Alizarin Red staining, cells were fixed for 5 min in 70% ethanol. The cells were stained with Alizarin Red solution . Stained cells were dehydrated in pure acetone, fixed in acetone-xylene (1 : 1) solution, and cleared with xylene. Differentiation on the uncoated glass surface was defined as the control group. The differentiated cells were analyzed by Western blotting with osteocalcin antibody  and 1% penicillin/streptomycin for 3 weeks. The medium was refreshed every 2–4 days. Intracellular lipid droplets indicating adipogenic differentiation, which was analyzed by Oil Red O staining  and confirmed by Western blotting using the antibody for adiponectin  supplemented with 10% FBS, 1% penicillin/streptomycin, 17.5 mM glucose, 25 ng/ml hEGF , 10 mM nicotinamide (Sigma-Aldrich), 2 nM activin-A (Sigma-Aldrich), 10 nM exendin-4 (Sigma-Aldrich), 100 pM hHGF , and 10 nM pentagastrin (Sigma-Aldrich). Differentiation was assessed by gene expression, Western blotting, and immune staining  and the reducing agent (Bolt Sample Reducing Agent), according to the instructions provided by the supplier. The protein mix was centrifuged and denatured at 70°C for 10 min. Then, the mixture was loaded on ready-to-use 4–12% Bis-Tris Plus mini-gels (Thermo Scientific). The gels were run at 200 V constant for 30 min using MES-SDS running buffer (Thermo Scientific). The iBlot2 dry blotting system was used to blot the proteins on the nitrocellulose membrane. iBind western processing device (Thermo Scientific) was used to stain the membrane. After incubation of the membrane with primary and secondary antibodies  on the culture flask, centrifuged at 14,000t-test in conjunction with the Newman–Keuls test and analysis of variance for repeated measures where appropriate. The significance of the results was calculated by SPSS 10.0 . Differences between the experimental and control groups were regarded as statistically significant when p < 0.05.All experiments were repeated at least three times. Data were analyzed using unpaired Student's The effect of modified collagen on cell morphology and proliferation was analyzed by F-actin staining with phalloidin and WST-1 assay, respectively . The phaBoth the expression of apoptotic genes and the active Caspase 3 staining were used to evaluate the apoptosis in the PI-MSCs cultured on the modified collagens , but no κB expressions were not changed significantly in the groups of glycated collagens. The expression of RAGE was slightly increased in all groups except for the cells on Col-A  cell markers in PI-MSCs was estimated relative to the expression in the cells differentiated on Col . The celμg/ml collagen while the concentration of AGE-products varied between 100 and 400 μg/ml in other studies. At low concentrations, the proliferation of mesangial cells was supported whereas it was inhibited at high AGE concentrations [Collagen was modified by reducing monosaccharides in this study to imitate the changes, which spontaneously take place with aging and cause age-related atrophic changes in tissues. To observe the diverse effect of modifications, collagen was treated with different types of monosaccharides beside the glucose. The actin structures of cells cultured on modified collagens showed distinct variations in the organization depend on the type of monosaccharide. While mannose- and arabinose-conjugated collagens supported the intactness of the actin structures in PI-MSCs during the culture, F-actins became tenuous, thinner, and abnormally distributed in Col-G and Col-R groups, but neither decrease in cell proliferation nor induction of apoptosis was observed in any group. Despite the number of attached cells on Col-R was significantly low, it did not affect the stability of the cell culture. Although other studies indicated that AGEs inhibited the proliferation of MSCs and reduced the cell numbers , this watrations , 13. SimκB was induced by TLR2 and TLR4 [The apoptosis was not observed in the cell culture. TLR-mediated signaling induced by glycated products was reported responsible for apoptosis signaling, and the expression of RAGE and NF-and TLR4 . Howeverand TLR4 . The weaand TLR4 . TLRs arand TLR4 . Similarand TLR4 . Beside and TLR4  without and TLR4 . Howeverand TLR4 . TLR2 anThe expression of RAGE in human MSCs was demonstrated to cause loss of their differentiation ability . TLR andIn the endocrine cell differentiation medium, PI-MSCs expressed insulin and Pdx1 at varying levels depending on the coated surface on which they were cultured. In the previous study with PI-MSCs, they were shown to differentiate into insulin-producing cells in the defined chemical cocktail , 25. By The studies related to the RAGE were shown that the maintenance mechanism of BM-MSCs could be disrupted during the stimulation of RAGE signaling . The RAGThe modification of collagen by glycation at low reducing sugar concentrations made it possible to improve the characteristics of the matrix polymer in favor of stem cell differentiation. By the modification of natural polymers, it could be possible to provide new features to the matrix proteins. Thereby, it might be possible to control the differentiation direction of the stem cells and to improve in generating functional mature cells. The modification of collagen by glycation is an irreversible process and has the chronic effect on tissue function and maintenance. It was found that the glycated collagen with glucose might have a deleterious effect, but the other reducible sugar acted differently, which should be analyzed in details.


The presented SERS sensor was also tested to detect a thiram at the micromolar level. We expect that our proposed AuNPs@GO/h-paper-based SERS sensor could be applied to point-of-care diagnostics applications in daily life and in spacecraft.Paper-based surface-enhanced Raman scattering (SERS) sensors have garnered much attention in the past decade owing to their ubiquity, ease of fabrication, and environmentally friendly substrate. The main drawbacks of a paper substrate for a SERS sensor are its high porosity, inherent hygroscopic nature, and hydrophilic surface property, which reduce the sensitivity and reproducibility of the SERS sensor. Here, we propose a simple, quick, convenient, and economical method for hydrophilic to hydrophobic surface modification of paper, while enhancing its mechanical and moisture-resistant properties. The hydrophobic paper (h-paper) was obtained by spin-coating diluted polydimethylsiloxane (PDMS) solution onto the filter paper, resulting in h-paper with an increased contact angle of up to ≈130°. To complete the h-paper-based SERS substrate, gold nanoparticles arranged on graphene oxide (AuNPs@GO) were synthesized using UV photoreduction, followed by drop-casting of AuNPs@GO solution on the h-paper substrate. The enhancement of the SERS signal was then assessed by attaching a rhodamine 6G (R6G) molecule as a Raman probe material to the h-paper-based SERS substrate. The limit of detection was 10 nM with an R The dom11 . Anotherructures . Generalructures .Recently, flexible SERS sensors have garnered much research attention owing to their capability for in situ and onsite detection, thereby avoiding the complicated extractions of analytes and tedious sample preparation steps ,12,13,142 of 0.966. We also investigated the presented SERS sensor to detect a thiram at the micromolar level.In this study, we propose a simple, convenient, timesaving, and economical method for hydrophobic surface modification of the filter paper and demonstrate the h-paper-based SERS sensor composed of gold nanoparticles arranged on graphene oxide (AuNPs@GO) flakes on the h-paper SERS substrate. The h-paper was prepared by spin-coating diluted polydimethylsiloxane (PDMS) on the filter paper, and the contact angle of the h-paper increased up to ≈130°. To fabricate a stable metal-nanoparticle platform, graphene oxide (GO) was used as a support for the arrangement of AuNPs. Graphene and graphene-based substrates are considered a promising material for SERS sensors owing to their large surface area and excellent molecule adsorption ability, and therefore, a number of graphene-based SERS devices have been proposed ,24,25,264, 99.99%), rhodamine 6G (R6G), and thiram were purchased from Sigma-Aldrich Korea, Seoul, Korea. Graphene oxide solution  was purchased from Graphene Square, Suwon, Korea. Filter paper was purchased from Whatman, Piscataway, NJ, USA. All reagents were used without further purification.Polydimethylsiloxane  was purchased from Dow Corning, Midland, MI, USA. Heptane , gold (III) chloride solution (HAuCl4 solution (2 M methanol-deionized water solution) was prepared, and approximately 1 mg/mL of GO solution was dispersed in 20 mL of 5 mM HAuCl4 solution; the mixture was then sonicated for 10 min. A UV lamp  with a central wavelength of 254 nm was then used to irradiate the above mixture for 60 min under magnetic stirring at 400 rpm. The suspension was subsequently centrifuged for 20 min at 10,000 rpm. The supernatant was discarded, and the sediments were redispersed in Milli-Q water. This process was repeated three times, and finally, 500 µL of the AuNPs@GO solution was prepared.A photoreduction technique was used to arrange the AuNPs on the surfaces of the GO flakes using UV light irradiation, as reported in a previous work . BrieflyPDMS with a 10:1 ratio of base to crosslinker by mass was prepared. The PDMS solution was diluted with heptane at different weight fractions , and the diluted PDMS solution was sonicated for 10 min to remove air bubbles. The h-paper was fabricated by spin-coating the diluted PDMS solutions on filter paper at 1000 rpm for 1 min. The PDMS was cured at room temperature for 48 h. The h-paper-based SERS substrate was completed by drop-casting 50 µL of the AuNPs@GO solution on the h-paper substrate. To evaluate the enhancement of the Raman scattering signal for the proposed SERS substrates, the AuNPs@GO/h-paper substrates were soaked in R6G in deionized (DI) water and thiram in ethanol of varying concentrations for 1 h. SERS measurements were performed after complete removal of the solvents by blowing nitrogen gas.The morphological and chemical compositions of the h-paper-based SERS substrates were characterized using scanning electron microscopy , transmission electron microscopy/energy dispersive spectroscopy , X-ray photoelectron spectroscopy , and UV-Vis spectroscopy . Raman spectra were measured using Raman spectroscopy . Raman spectra of R6G molecules were obtained with a laser excitation wavelength of 532 nm, a laser power of 2 mW, an acquisition time of 10 s, and an accumulation of 5. For thiram molecules, Raman spectra were obtained in a laser excitation wavelength of 785 nm, a laser power of 2.4 mW, an acquisition time of 10 s, and an accumulation of 5. The contact angles were characterized using a homemade contact angle measurement system and ImageJ 1.52h version  .4 solution mixed with 2 M methanol-DI water under UV radiation for 60 min, as shown in 2OH under UV light, thus accelerating the reduction of AuC4− to Au0 on the GO surfaces. The schematic representation of the fabrication process for the h-paper-based SERS substrate is presented in 2, as shown in 7/2 peak was observed at a binding energy of 82.9 eV, and the Au 4f5/2 peak was observed at 86.5 eV, indicating the binding of AuNPs to the surface of GO [To fabricate the h-paper-based SERS substrate, about 50 µL of the AuNPs@GO solution was drop-casted onto the h-paper substrate and dried at ambient conditions. ce of GO .−3 to 10−8 M. The R6G spectra at 10−3 M on the AuNPs@GO substrate exhibited strong peaks of the vibrational bands at about 612, 773, 1134, 1310, 1363, and 1651 cm−1, corresponding to the Raman characteristic peaks of R6G  was about 14.2%. To evaluate the reproducibility of the proposed AuNPs@GO/h-paper SERS substrates, we fabricated 15 AuNPs@GO/h-paper SERS substrates, as shown in −3 M. −1, and the corresponding RSD was about 11.3%. For a reliability test, we compared the sensitivity of AuNPs@GO/h-paper substrates that were kept for 0 and 15 days, as shown in −1 slightly decreased by about 5.4% compared to the initial state (day 0).s of R6G , as show−1 for thiram at 10−3 M [−1, which was attributed to a CH3 deformation vibration and C–N stretching vibration, was selected to evaluate the sensitivity of AuNPs@GO/h-paper substrates. −3 to 10−6 M. −1 as a function of the logarithmic concentration of thiram on the AuNPs@GO. The LOD for thiram was 1 μM.To demonstrate the possibility of practical utilization, we investigated the SERS activity of AuNPs@GO/h-paper substrates with thiram, which is a representative fungicide used for the protection of fruits and vegetables . Figure t 10−3 M . The str2 of 0.966. To demonstrate the possibility of practical utilization, the presented SERS sensor was tested to detect a thiram at the micromolar level. We expect that our proposed AuNPs@GO/h-paper-based SERS sensor could be applied for point-of-care diagnostics applications in daily life and in spacecraft.In summary, we developed a simple, convenient, timesaving, and economical method for hydrophobic surface modification of the filter paper and demonstrated the AuNPs@GO/h-paper-based SERS sensor. Hydrophobic treatment increased the contact angle and decreased the contact area of the aqueous solution. These brought about an extended retention time of the AuNPs@GO solution in the reduced contact area, resulting in the concentrated AuNPs@GO on h-paper, enabling the creation of SERS hot-spots. The sensitivity was measured using R6G molecules with concentrations from 1 mM to 10 nM as Raman probe materials. The limit of detection (LOD) was 10 nM with an R


In this study, double-walled microspheres made of glucose core, hydroxyl-terminated poly(lactide-co-glycolide) (Glu-PLGA), and carboxyl-terminated PLGA were fabricated using a modified water-in-oil-in-oil-in-water (w1/o/o/w2) emulsion solvent evaporation technique for the controlled release of a model protein, lysozyme. Microspheres size, morphology, encapsulation efficiency, lysozyme in vitro release profiles, bioactivity, and structural integrity, were evaluated. Scanning electron microscopy (SEM) images revealed that double-walled microspheres comprising of Glu-PLGA and PLGA with a mass ratio of 1:1 have a spherical shape and smooth surfaces. A statistically significant increase in the encapsulation efficiency (82.52 ± 3.28%) was achieved when 1% (w/v) polyvinyl alcohol (PVA) and 2.5% (w/v) trehalose were incorporated in the internal and external aqueous phase, respectively, during emulsification. Double-walled microspheres prepared together with excipients  showed a better control release of lysozyme. The released lysozyme was fully bioactive, and its structural integrity was slightly affected during microspheres fabrication and in vitro release studies. Therefore, double-walled microspheres made of Glu-PLGA and PLGA together with excipients  provide a controlled and sustained release for lysozyme.Double-walled microspheres based on poly(lactide- Single-walled microspheres comprising of Glu-PLGA and PLGA were also prepared to show the effectiveness of blending of two different PLGAs in reducing the high initial burst. Moreover, double-walled microspheres were prepared together with excipients  to investigate the phenomena of lysozyme denaturation during microspheres fabrication.In recent years, controlled-release formulations of the therapeutic protein and peptide drugs using biodegradable polymeric devices have attracted much attention in research community around the world ,2,3,4. Mdelivery . PLGA andelivery . Due to delivery ,11,12. Idelivery ,14,15. Idelivery ,17. Manydelivery , fluidizdelivery , spray ddelivery , a precidelivery ,22, and delivery ,24,25. Bde drugs ,24. In tco-glycolide)  (Glu-PLGA) average Mn 15,000 and 50,000, polyvinyl alcohol , chicken egg-white lysozyme, bicinchoninic acid kit (BCA1-1KT), micrococcus lysodeikticus (ATCC 4698), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich . Ethyl acetate (EA) and dichloromethane (DCM) were purchased from Fisher Scientific . PLGA (50:50 with an intrinsic viscosity 0.4 dL/g) were obtained from Purac . All of the other chemicals and solvents used in this study were of analytical grade.Poly(lactide-1/o/o/w2) emulsion solvent evaporation method was modified and employed to fabricate lysozyme encapsulated in double-walled microspheres [® T 10 . The obtained water-in-oil (w1/o) emulsion was then poured into dichloromethane (DCM) solution of Glu-PLGA. The resulting two phases were homogenized using the same homogenizer. The water-in-oil-in-oil (w1/o/o) emulsion was formed which was subsequently transferred to an external aqueous phase containing 1% (w/v) PVA solution. In this step, water-in-oil-in-oil-in-water (w1/o/o/w2) emulsion was formed after homogenization using the same homogenizer. The resultant w1/o/o/w2 emulsion was then added to an aqueous 0.5% (w/v) PVA solution and stirred magnetically at room temperature and pressure was reduced to evaporate dichloromethane and ethyl acetate. The hardened microspheres in suspension were then centrifuged and washed with double distilled water to remove the excess PVA. Finally, the microspheres were freeze-dried overnight using a freeze-dryer  and stored at −20 °C until further characterization. In addition, conventional water-in-oil-in-water (w1/o/w2) emulsion solvent evaporation technique was used to fabricate single-walled microspheres of Glu-PLGA and PLGA for comparison. A previously developed water-in-oil-in-oil-in-water . Particle size distribution (polydispersity) was expressed by the span value, which is calculated as follows:D10%, D50%, and D90% are the particle diameters at 10%, 50%, and 90% cumulative volume, respectively, and the cumulative percentage (%) is computed by summing the percentages of particles smaller than their equivalent diameter.The particle size of the hardened microspheres was determined by laser diffraction using a Laser Particle Size Analyzer BT-9300H . Prior to freeze-drying, an aliquot of the wet microspheres was employed to measure the particle size, which is used to estimatethe volume median diameter  was used to observe the shape and surface morphologies of the microspheres. Freeze-dried microspheres were first mounted onto metal stubs using double-sided adhesive tape. The stubs were then coated with a thin layer of gold under vacuum using a sputter coater. The microspheres were imaged using SEM with an accelerating voltage set to 5–20 KV.A Scanning Electron Microscope (SEM)  was employed to explore the core and shell polymer layer of double-walled microspheres comprising Glu-PLGA and PLGA . In thisw/v) SDS (sodium dodecyl sulfate). A micro-BCA protein assay kit was used to quantify lysozyme in the clear solution. In this method, empty microspheres (lysozyme free) were also used as a control. The following equations were used to estimate lysozyme loading and encapsulation efficiency.In this study, micro-BCA protein assay method developed by Sah (1997) was used to quantify lysozyme in the microspheres . Freeze-At the end of microspheres fabrication, lyophilized microspheres were weighed and the yield was measured by:3) aqueous solution  was added in the tubes as a bacteriostatic agent. The tubes were placed in an incubator shaker and agitated horizontally at 100 rpm with 37 °C. At specific time intervals, the tubes were centrifuged and the supernatant was withdrawn completely. A constant pH sink condition was maintained throughout the release studies as an equal volume of fresh buffer was poured after each sampling. A micro-BCA protein assay kit was used to quantify the lysozyme level in the supernatant. Each of the samples was analyzed in triplicate.To study the in vitro release profiles, approximately 10 mg lyophilized microspheres were suspended in 1.5 mL phosphate buffer saline (pH 7.4) containing Eppendorf tubes. Sodium azide . For measuring the turbimetric rate, 10 µL aliquot of the day 1 released lysozyme sample (concentration adjusted to 50 µg/mL) was taken in a cuvette and mixed with 1.3 mL substrate suspension. The cuvette was immediately placed in a spectrophotometer . The decrease in the turbidity of the suspension was measured for 3 min at 450 nm. The following equation was used to calculate the bioactivity of released lysozyme [Since lysozyme hydrolyses the bacterial cell walls, lysozyme ,30.w/v) in internal aqueous phase and 2.5% trehalose (w/v) in the external aqueous phase was examined. The extracted and released lysozyme was mixed with a sample buffer . The buffer to sample ratio of mixing was maintained as 1:4 (v/v). The samples were warmed for 5 min at 95 °C prior to loading. The process of electrophoresis was performed at a constant voltage of 200 V in a running buffer (0.3% (w/v) Tris base, 1.4% (w/v) glycine, 0.1% (w/v) SDS, pH 8.3) using a Bio-Red Mini-Protean electrophoresis system. The gels were stained with Coomassie blue to visualize the protein bands. The molecular weights of the visualized bands were compared to a protein molecular weight marker . Native lysozyme treated under the same condition was used as a control.A non-reducing SDS-PAGE was performed to determine the possible degradation or aggregation of lysozyme during microspheres fabrication and in vitro release studies. In this case, lysozyme encapsulated microspheres as prepared in presence of 1% PVA  were used to assess the statistical difference. A statistically significant difference was considered when the p value was less than 0.05.The results were expressed as the mean value ± standard deviation for three separate measurements. One-way analysis of variance (ANOVA), followed by p < 0.05) is consistent with the previous results [p < 0.05) was observed in formulations LF1 and LF3 as compared to other double-walled and single-walled formulations. Polymer viscosity has a great influence on the mean size and span value of microspheres. The viscosity of polymer solution increases with increasing mass ratio of a polymer mixture, which resists emulsion droplets to be broken down into smaller droplets using the same shear stress [Microspheres mean particle size and size distributions  are shown in  results ,27. Morer stress ,27. TherThe surface morphologies (shape and size) of lysozyme loaded double-walled and single-walled microspheres were studied by SEM. The SEM analysis demonstrated that the double-walled microspheres made of Glu-PLGA and PLGA in a mass ratio of 1:1 were spherical in shape, smooth, and non-porous surfaces a,c. In cThe polymer (Glu-PLGA and PLGA) orientation in prepared microspheres was evaluated using a selective dissolution technique, as described in the previous publications, and found Glu-PLGA and PLGA as the core and shell layer, respectively ,27.p < 0.05) in the encapsulation efficiency of formulations LF2 and LF4 is assumed to be due to the increase in polymer viscosity with an increasing polymer mass. High viscous polymer-solution reduces the mobility of proteins in the emulsion droplets. Thus, the diffusion of hydrophilic lysozyme into the continuous aqueous phase might be reduced, which results in a higher encapsulation efficiency. Moreover, the polymer precipitation rate is faster at a higher polymer concentration, which in turn could slow down the diffusion rate of lysozyme from the emulsion droplets to the continuous phase, as the diffusion time is very short. Therefore, the increasing of polymer mass is considered to be the reason of slightly higher encapsulation efficiencies of formulations LF2 and LF4.The encapsulation efficiency and actual loading of lysozyme in double-walled and single-walled microspheres are shown in p < 0.05) in encapsulation efficiency when compared to double-walled formulations. SEM images of single-walled and double-walled formulations revealed that single-walled formulations were highly porous, whereas double-walled formulations were non-porous. The rapid diffusion of hydrophilic lysozyme from the disperse phase to the continuous phase is accounted for the formation of these pores. Therefore, single-walled formulations could not retard the rapid diffusion of lysozyme into the continuous phase, which resulted in low encapsulation efficiency. Although the same amount of polymer corresponding to formulations LF1 and LF3 was used in formulation LF6 , the encapsulation efficiency of LF6 was not as high as LF1 and LF3. Lysozyme molecules encapsulated in the core of double-walled microspheres might be shielded from the rapid diffusion to the surrounding continuous phase due to the presence of another outer polymer layer. Single-walled microspheres cannot suppress the rapid diffusion of hydrophilic lysozyme to the external aqueous phase during emulsification and hardening, as they do not have any core/shell layer. Hence, lysozyme loaded double-walled microspheres showed a higher encapsulation efficiency as compared to single-walled microspheres.The encapsulation efficiencies of single-walled microspheres prepared in formulations LF5, LF6, LF7, and LF8 were calculated as 51.67 ± 3.56%, 62.03 ± 4.15%, 53.88 ± 2.97%, and 52.23 ± 3.90%, respectively. These single-walled formulations showed a statistically significant decrease  SDS containing 0.05 M NaOH solutions. The protein content in the DMSO digested solution was then quantified by a micro BCA protein assay kit. At the same time, the enzymatic activity of the released lysozyme was determined.To investigate the incomplete release above, the remaining insoluble residues present in the micro centrifuge tubes after 70 days in vitro release studies were centrifuged, washed three times with distilled water, and freeze-dried over night. The freeze-dried residues were then dissolved in DMSO and 0.5%  solution was used as an internal aqueous phase (w1), whereas 2.5% aqueous trehalose (w/v) solution was used for the preparation of an external aqueous phase (w2). The formulation and process variables were the same as double-walled microspheres were prepared in formulation LF1.In these circumstances, attempts were made to improve the in vitro release profiles of lysozyme from double-walled microspheres by the incorporation of PVA in the internal aqueous phase and trehalose in the external aqueous phase. Lysozyme dissolved in 1% aqueous PVA (w/v) in the internal aqueous phase did not show a marked difference in the mean particle size as compared to microspheres prepared free from PVA in the internal aqueous phase. In contrast, a statistically significant increase (p < 0.05) in the mean particle size was observed when microspheres were prepared with the incorporation of 2.5% trehalose (w/v) in the internal aqueous phase. With the addition of trehalose in the internal aqueous phase, the w1/o emulsion droplets might be destabilized due to the inward diffusion of water molecules from the external to the internal aqueous phase because of high osmotic pressure. This phenomenon leads to swelling and disrupting of the internal emulsion droplets, which favors coalescence of the micro droplets, and results in the formation of microspheres with a slightly larger size. It has been reported that the presence of highly water soluble excipients  in the internal aqueous phase at higher concentrations forms highly porous and even ruptured microspheres [w/v) in the internal aqueous phase and 2.5% trehalose (w/v) in the external aqueous phase interestingly reduced the microspheres size to 2.61 ± 0.50 µm as compared to the addition of trehalose in the internal aqueous phase alone. This might be due to the increase in viscosity of the external aqueous phase as trehalose was incorporated in the external continuous phase. The increased viscosity of the external aqueous phase might inhibit the diffusion of the external aqueous phase to internal aqueous phase and resulting in a balancing of osmotic pressure between the two phases. This balancing of osmotic pressure could stabilize the emulsion droplets against coalescence and result in reduced microspheres size. In a study, the addition of glucose in the external aqueous phase showed a remarkable decrease in the microspheres size from 200 µm to 45 um [w/v) in the external aqueous phase reduced the particle size to 85 µm and a gradual decrease in the microspheres size was observed with increasing the concentration of glucose solution. In the above studies, the increased viscosity of the external aqueous phase due to the addition of glucose was considered to be a reason of producing microspheres with a reduced particle size.ospheres . It was to 45 um . The avew/v) in the internal aqueous phase. In contrast, the external morphologies of double-walled microspheres prepared with 1% PVA (w/v) in internal aqueous phase and 2.5% (w/v) trehalose in the external aqueous were spherical in shape and had smooth and nonporous surfaces (w/v) trehalose in the internal aqueous phase leads to an inward diffusion of the external aqueous phase to internal aqueous phase for balancing of the osmotic pressure between the two phases, which resulted in swelling and disrupting of the internal droplets and a loss of drug molecules being encapsulated. The water diffusion channels might appear as pores after solidification of the microspheres. Therefore, the porous and squeezed surfaces of the microspheres prepared with 2.5% trehalose (w/v) in the internal aqueous phase might be a result of the inward diffusion of water molecules from the external to the internal aqueous phase. The water diffusion might be inhibited due to the incorporation of 1% PVA (w/v) in the internal aqueous phase and 2.5% trehalose (w/v) in the external aqueous because of the balancing of osmotic pressure, which resulted in spherical, dense, and nonporous microspheres. surfaces c,d. As dw/v) solution alone in the internal aqueous phase showed a statistically significant decrease (p < 0.05) in encapsulation efficiency when compared to other excipients containing formulations  in the internal aqueous phase and 2.5% trehalose (w/v) in the external aqueous phase, no water molecules might diffuse from the external to the internal phase, which contributes to the formation of spherical, dense, and absolutely nonporous and compact shape microspheres, and result in a high encapsulation efficiency.The incorporation of 2.5% trehalose  in the internal and external aqueous phase are shown in w/v) in the internal aqueous phase slightly reduced the day 1 release to 17.32 ± 3.84%, whereas the cumulative release was increased to 55.97 ± 4.15% after day 70. Among the excipients-containing formulations, a more sustained release of lysozyme was achieved with the incorporation of 1% PVA (w/v) in the internal aqueous phase and 2.5% trehalose (w/v) in the outer aqueous phase during microspheres preparation. In this case, a tri-phasic release profile was observed. Initially, 24.32 ± 3.57% of lysozyme was released within the first 24 h, then the release was continued very slowly until day 14, and subsequently a faster and sustained release was observed until day 70. Between day 1 and 70, the highest amount of 39% lysozyme was released from the microspheres prepared with 1% PVA (w/v) in the internal aqueous phase and 2.5% trehalose (w/v) in the external aqueous phase. This might be due to the presence of PVA in the internal aqueous phase that could stabilize lysozyme against aqueous/organic interface induced denaturation and aggregation. The incorporation of PVA in the internal aqueous phase improves lysozyme stability probably by competing with lysozyme adsorption at large water/organic interface [The incorporation of 1% PVA . The incorporation of 1% PVA (w/v) in the internal aqueous phase and 2.5% (w/v) trehalose in the external aqueous phase showed approximately 24% of lysozyme release from double-walled microspheres after day 1. The release continued in a sustained manner that reached up to approximately 64% after day 70. Therefore, double-walled microspheres comprising of Glu-PLGA and PLGA provided an improved lysozyme release profiles when excipients  were co-encapsulated during microspheres fabrication.In this study, a model protein-lysozyme encapsulated in double-walled microspheres comprising of Glu-PLGA and PLGA has been fabricated successfully using a modified w


Increased expression of p75 receptor with decreased expression of pro-BDNF was observed after chronic PCPA. Seven-day treatment with 8-OH-DPAT reestablished the expression of pro-BDNF modified by PCPA, and induced an increase in the expression of p75 receptor. It has been demonstrated that PCPA-treated mice have higher number of immature neurons in the HC. Given that immature neurons participate in the pattern separation process, the object pattern separation test was conducted. A better performance of hyposerotonergic mice was not confirmed in this assay. Altogether, our results show that molecules in the BDNF signaling pathway are differentially expressed under diverse configurations of the serotonergic system, allowing for fine-tuning of the neurogenic process.Neurotrophic factors are relevant regulators of the neurogenic process at different levels. In particular, the brain-derived neurotrophic factor, BDNF, is highly expressed in the hippocampus (HC) of rodents and participates in the control of neuronal proliferation, and survival in the dentate gyrus (DG). Likewise, serotonin is also involved in the regulation of neurogenesis, though its role is apparently more complex. Indeed, both enhancement of serotonin neurotransmission as well as serotonin depletion, paradoxically increase neuronal survival in the HC of mice. In this study, we analyzed the protein expression of the BDNF isoforms, i.e., pro- and mature-BDNF, and their respective receptors p75 and TrkB, in the HC of mice chronically treated with para-chloro-phenyl-alanine (PCPA), an inhibitor of serotonin synthesis. The same analysis was conducted in hyposerotonergic mice with concomitant administration of the 5-HT With the extension of life expectancy, several pathologies affecting the central nervous system have gained greater visibility. In this context, disentangling the etiopathogenesis of neurodegenerative diseases and seeking for more effective therapies represent key challenges. Strategies to replace lost neurons because of neurodegenerative processes are focused on two types of interventions: on the one hand, transplantation of exogenous nerve cells and, on the other, the use of the endogenous neural niches still active in the adult nervous system . Indeed,1A receptor for 7 days was enough to reestablish the increased survival of 1-week-old neurons shown in hyposerotonergic mice, induced either by genetic, or pharmacological ablation . Three-four week-old mice, bred in barrier-conditions to maintain an SPF status, were transported in environmentally controlled conditions to our institute’s animal facility. After arrival, mice were housed in 1284L Eurostandard Type II Long (365 mm × 207 mm × 140 mm) Tecniplast microisolator cages with filter tops , with autoclavated aspen shavings as bedding and tissue paper as nesting material. Mice were maintained under controlled conditions, i.e., 22 ± 2°C room temperature, 60% relative humidity, 12–12 h light–dark cycle (lights on at 8 a.m.), pelleted food for rodents (Cooperación) and water Serotonin depletion in mice was induced by oral administration of para-chloro-phenyl-alanine (PCPA), an inhibitor of the rate limiting enzime tryptophane hydroxylase, during 8 weeks, as previously described . Brieflyn = 6) or vehicle (n = 5) administration, mice were killed by cervical dislocation and both HC were obtained. Tissue was homogenized with 250 μl of RIPA buffer along with protease inhibitors  and centrifuged at 4°C for 30 min at 13000 (r/min). The supernatants were recovered and proteins levels were quantified by the Bradford protein assay. Samples (50 μgr in 5× loading buffer) were then loaded into SDS-PAGE gels (12 or 15%) and transferred onto nitrocellulose membranes using the Mini-PROTEAN® Tetra System (BIO-RAD) for 1 h.Chronic PCPA treatment during 8 weeks increases survival of 4 week-old neurons in the HC . To stud® Molecular Imager. Densitometry was carried out using ImageJ software (Membranes were incubated for 1 h with blocking solution (5% milk in TBST) and then probed overnight at 4°C with mouse anti-BDNF , rabbit anti-p75 , rabbit anti-TrkB , and rabbit anti-proBDNF  in TBST. β-III Tubulin was used as a loading control . Binding of primary antibodies was visualized with anti-mouse HRP-conjugated secondary antibody  or anti-rabbit HRP-conjugated secondary antibody . Membranes were developed using the ECL Plus Western blotting substrate (Thermo Fisher Scientific) for chemifluorescence with the Stormsoftware . The sig1A receptor for 7 days is able to reestablish the basal level of survival of 1 week-old newborn neurons in the HC of PCPA-chronically treated mice (1A receptor agonist 8-Hydroxy-2-(di-n- propylamino)tetralin (8-OH-DPAT) was administered to a group of PCPA- (n = 11) or vehicle- (n = 12) treated mice. Mice received palatable cubes with PCPA or vehicle for 4 weeks, as explained above. On the 5th week, animals continued receiving PCPA or vehicle, and simultaneously received two daily intraperitoneal (i.p.) injections of 8-OH-DPAT at 0.5 mg/kg or vehicle (NaCl 0.9%) for 7 days as previously described  and habituated for 3 days to the open field without any objects. They were placed in the arena and left for 10 min to explore. Mice were then tested 48 hs after the last habituation session. First, in a pre-test session, they were presented two identical objects placed in two symmetrical spots in the center of the arena. The objects were caramel color bottles of 7 cm of height filled with a dark solution so that mice were not able to move them. Animals were left 4 min to explore. The test session was performed 1 h later, where one of the objects was placed in a novel location inside the arena 2, 4, 6, or 8 cm away from the initial location . Position 1 meant no change in any object. Each group was assigned a different position. Mice were left again 4 min to explore. Time spent exploring each object in both sessions was recorded. Exploration was defined as directing the nose toward the object at a distance of no more than 2 cm and/or touching the object with the nose. Sitting on the object was not considered as exploratory behavior. Mice that spent less than 10 s in total exploratory time in the pre-test or test were eliminated from the data, since it has been shown that animals require at least 10 s of object interaction for reliable object discrimination  test in our experimental conditions, the protocol described by mination . Exploran = 13–15), and received a palatable cube containing either PCPA or vehicle during 8 weeks, as described above. PCPA was administered as a tool to increase the survival of newborn neurons in the HC : Sigma, C6506; 8-OH-DPAT (8-Hydroxy-2-(dipropylamino) tetralin hydrobromide): Sigma, H8520.t-test. Results in Experiment 2 were analyzed by a two-way analysis of variance (ANOVA), with hyposerotonergic condition, and DPAT treatment as factors. Tukey’s test was used for post hoc comparisons. Simple effects tests were performed to analyze differences inside each condition. The OPS was analyzed using the one-sample t-statistics in order to assess whether the discrimination index, for each experimental condition separately, differed significantly from 0. In all cases, P < 0.05 was considered statistically significant. A summary of tests employed and statistics is presented in All data were checked to verify whether normality and homoscedasticity assumptions were met. Different treatments in Experiment 1 were analyzed using Student’s p = 0.0427) in PCPA-treated mice.Protein-expression of BDNF isoforms and their receptors was analyzed in the HC of 8-week PCPA- or vehicle-treated mice . Whereas1A agonist DPAT or NaCl 0.9% (p = 0.0085), and a tendency to increased expression of p75 receptor (p = 0.0931), without significantly modifying the expression of mBDNF or TrkB receptor. One week administration of DPAT in vehicle-treated mice induced a significant decrease in the expression of mBDNF (p = 0.0403) and TrkB receptor (p = 0.0004), without significantly affecting the levels of proBDNF or p75 receptor. Finally, administration of DPAT to PCPA-treated mice induced a significant increase in the expression of proBDNF (p = 0.0035) and p75 receptor (p = 0.0239), without significantly modifying the levels of mBDNF and TrkB receptor.Protein expression of the BDNF isoforms and their receptors was analyzed in the HC of PCPA- and vehicle- chronically treated mice receiving the 5-HTaCl 0.9% . StatistaCl 0.9% . Five wep = 0.0088), whereas P3 showed a tendency for significance (p = 0.1203). Altogether, as naïve mice are barely able to discriminate positions in P3, the performance in this position can be improved, which would result in a higher index and possibly, a p value <0.05. If mice were tested in P5, where the discrimination index is high, a difference between treated and control mice would not be seen, as all of them are expected to perform well.In the set up, each mouse was assigned to a different position for the moved object, identified as P1, P2, P3, P4, or P5 . As the p < 0.05). No significant difference in locomotor activity was seen between mice treated with PCPA and vehicle. After a resting day, mice were presented with two identical bottles for 4 min at P1. One hour later, one of the bottles was moved to P3 and the animals were left another 4 min to explore. Five mice were eliminated from the data because they did not meet the criteria for minimal exploration time of 10 s. A discrimination index was calculated, as previously explained (p = 0.0435), PCPA-treated mice showed a high p value (p = 0.6736).To study the role of newborn cells in the HC of mice treated with PCPA or vehicle, animals were assayed in the OPS at P3, given that subtle differences, i.e., a mild better performance, between experimental groups were expected. Mice were habituated to the open field for 3 consecutive days, for 10 min each day. All mice got habituated to the arena , with lexplained . WhereasPrevious reports on hyposerotonergic mice demonstrated increased survival of the newborn neurons generated in the adult HC, with unchanged cell proliferation. The results presented herein support the hypothesis about an interplay between the BDNF signaling pathway in the HC and the serotonergic system that could participate in the regulation of the survival of newborn neurons. On the other hand, in our experimental conditions, a facilitating role for supernumerary immature neurons on pattern separation assays could not be confirmed.–/– .RF designed the study, conducted the experiments, and wrote the manuscript. KS, AB, and AR conducted the experiments. SD designed the study, supervised the experiments, wrote the manuscript, and provided funding.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.